Methylmercury (MeHg) is a global pollutant and potent neurotoxin whose abundance in the food chain mandates additional studies on the consequences and mechanisms of its CNS toxicity. Formulation of our new hypotheses was predicated on our appreciation for (a) the remarkable affinity of mercurials for the anionic form of sulfhydryl (-SH) groups, (b) the essential role of thiols in protein biochemistry, and (c) the role of molecular chaperone proteins, such as heat shock protein 90 (Hsp90), in the regulation of protein redox status by facilitating the formation and breakage of disulfide bridges. Given the high affinity of MeHg for thiols, we hypothesized that MeHg interferes with Hsp90 chaperone function resulting in altered cellular homeostasis and neurotoxicity. Among the client proteins known to bind to Hsp90 are type I (neuronal) nitric oxide synthase (NOS), and cytosolic prostaglandin E synthase (cPGESIp23). Additionally, Hsp90 reduces the mitochondrial electron transport protein, cytochrome c (cyt c). The specific aims are to [1] demonstrate that MeHg physically binds to Hsp90, altering Hsp90 chaperone function. [2] Determine whether MeHg increases the activity of the cPLA2-cyclooxygenase-1 (COX-1)-PGES enzymatic pathway resulting in enhanced prostaglandin E2 (PGE2) production by altering the binding of Hsp90 to PGES/p23. [3] Demonstrate that by altering the association between Hsp90 and neuronal NOS (nNOS) MeHg uncouples nNOS activity, resulting in NOS-derived superoxide (-O2) production and reduced NO bioavailability. [4] Determine whether MeHg-induced oxidant stress occurs via inhibition of the mitochondrial electron transport chain, causing increased -O2 production and reduced levels of the intracellular antioxidant, glutathione. The experimental approach includes cell-free lysates, rat-derived in vitro astrocyte, neuron, and astrocyte/neuron co-cultures, as well as in vivo corroborative studies in the rat. MeHg-mediated alterations in Hsp90/client protein interactions offers an innovative and unifying mechanism integrating the known propensity of MeHg to form complexes with -SH-containing ligands and MeHg-induced oxidant stress, mitochondrial dysfunction and CNS toxicity. The results of these studies will shed new light on meaningful mechanisms of MeHg-induced neurotoxicity, and pave the way for new pharmacological modalities for treatment. Additionally, studies on altered post-translational modification of Hsp90 client proteins might offer new mechanistic insight into other neurodegenerative disorders, and therefore they have broad biological implications